CYTOSKELETAL DYNAMICS AT SYNAPSES: CHARACTERIZATION OF NOVEL MICROTUBULAR REGULATORS AND THEIR POTENTIAL ROLE IN RESILIENCE TO NEURODEGENERATION

In neurons, the microtubule (MT) cytoskeleton composed of α- and β-tubulin, plays a key role in the development and maintenance of axons and dendrites. At the synaptic compartment, dynamic microtubules enter into dendritic spines, the tiny protrusions that support the post-synaptic side of most excitatory synapses in the brain, contributing to synaptic maintenance and plasticity. Furthermore, recent studies in our laboratory demonstrated that in Alzheimer’s disease (AD) spines that were invaded by microtubules successfully resisted oAβ injury, whereas non-invaded neighbouring spines were frequently eliminated contributing to early decay of synaptic connections associated with memory failure. Based on these findings, we hypothesized that the entry of microtubules into the spines provides a direct route for the transport of selective synaptic components that may contribute to protect spines against amyloid toxicity. To identify both the cargo molecules that are being trafficked and the proteins that promote microtubule entry, we employed a proximity labeling assay using a split version of the biotin ligase TurboID. One fragment was linked to the microtubular protein EB3 and the other to the F-actin-binding peptide LifeAct, allowing enzyme reconstitution and biotinylation of nearby synaptic effectors specifically during microtubule entry into the spines. Biotinylated proteins will be then identified by mass spectrometry. This work will uncover key regulators and cargoes involved in microtubule entry into spines, shedding light on mechanisms of synaptic resilience and offering potential targets for early intervention in neurodegenerative diseases like AD.